CN104478993A - Non-protein amino acid antimicrobial peptide and application thereof - Google Patents

Abstract

The invention discloses a non-protein amino acid antimicrobial peptide, which has any one of the following sequence lists: SEQ ID NO.1: R-FPA-R-FPA-EtBn; SEQ ID NO.2: R- BTF-R-BTF-EtBn; SEQ ID NO. 3: R-FPA-R-EtBn; SEQ ID NO. 4: R-Nag-R-EtBn. The four antimicrobial peptides of the present invention have good structural specificity, strong resistance to enzymatic hydrolysis in vivo, clear action mechanism, small toxic and side effects, easy chemical synthesis, and potential to be used as a drug. The invention also discloses the application of the antibacterial peptide.

Description

A kind of non-protein amino acid antimicrobial peptide and its application

This application is a divisional application based on the Chinese invention patent application with application number 2013102466355.

technical field

The invention belongs to the field of antibacterial peptides, in particular to an artificially designed non-protein amino acid antibacterial peptide and its application.

Background technique

At present, the resistance to antimicrobial drugs is increasing day by day, especially the abuse of antibiotics is becoming more and more serious. Human beings urgently need to develop a new class of antibacterial drugs with high efficiency, low toxicity and no residue. The antimicrobial peptide antimicrobial peptide has attracted great attention for its unique biological activity and special mechanism of action different from traditional antibiotics. It has the advantages of broad antibacterial spectrum, good stability, small side effects, and no drug resistance. It is expected to overcome the current Antibacterial drugs have a series of problems such as strong drug resistance and large side effects. Scientists believe that it is expected to become a new generation of antibacterial, antiviral, and anticancer drugs.

Antibacterial peptides (ABP), also known as anti-microbacterial peptides (AMP), are the products of a series of immune responses produced by organisms against the invasion of external pathogenic substances, and can eliminate a class of mutant cells in the body. Biologically active small molecule polypeptides generally consist of 12-50 amino acid residues, contain net positive charges and more than 50% hydrophobic residues. Antimicrobial peptides can not only act on bacteria, fungi, protozoa, viruses and tumor cells, but also have the ability to enhance the body's immunity and accelerate wound healing. Antimicrobial peptides are more stable at higher ionic strength and lower or higher pH. At the same time, it has a faster bactericidal ability and is less likely to produce drug resistance. So far, no drug resistance caused by antimicrobial peptides has been found, and these natural antimicrobial peptides have no teratogenic effect and will not produce accumulation poisoning. At present, a variety of antimicrobial active peptide molecular design and modification methods have been used in the research of antimicrobial peptides. It is expected to improve the antibacterial activity while further improving its stability in plasma, reducing toxic and side effects, and laying the foundation for the clinical application of antimicrobial peptides.

Contents of the invention

The purpose of the present invention is to provide a non-protein amino acid antibacterial peptide, which introduces a design method of unnatural amino acids on the basis of the design concept of small molecule cationic antibacterial peptides. The peptide chain is shorter, and each polypeptide chain does not exceed 4 amino acid residues. , which makes the synthesis easy; high charge, each designed polypeptide has at least two positive charges; strong amphiphilicity ensures that it has low hemolytic toxicity; many dominant amino acids; and the end of the peptide chain is ethylaniline with good flexibility and strong hydrophobicity chemical protection. (At the same time, the high-throughput screening is realized through quantitative structure-activity relationship analysis, which saves the time and cost of novel antimicrobial peptide design and screening, and improves the rationality and feasibility of antimicrobial peptide molecular design.) The four items described in the present invention Antimicrobial peptides have good structural specificity, strong resistance to enzymatic hydrolysis in vivo, clear mechanism of action, less toxic and side effects, easy chemical synthesis, and potential for drug production. The invention also provides the application of the antibacterial peptide.

In order to achieve the above object, the present invention adopts following technical scheme:

A non-protein amino acid antibacterial peptide, the antibacterial peptide has any one of the sequences in the following sequence listing:

SEQ ID NO.1: R-FPA-R-FPA-EtBn;

SEQ ID NO.2: R-BTF-R-BTF-EtBn;

SEQ ID NO.3: R-FPA-R-EtBn;

SEQ ID NO. 4: R-Nag-R-EtBn.

Based on the design of the antimicrobial peptide library, considering the structure-activity relationship, dominant amino acids, dominant sites and secondary structure trends, it is found that in most cationic antimicrobial peptide sequences, the positively charged arginine R and the contribution of hydrophobic The dominant tryptophan is its most dominant amino acid. In this study, on the basis of the antibacterial hexapeptide, the sequences were further streamlined, and only these two optimal amino acids were retained. On this basis, 4 peptoids were finally screened out through the transformation and modification of unnatural amino acids.

The amidation treatment of the C-terminal of the antimicrobial peptide contributes to the increase of the static charge of the peptide and the stability of the antimicrobial peptide, enhances the cationicity of the peptidomimetic, and increases the antibacterial activity and stability of the antimicrobial peptide. There is no redundant residue at the N-terminal of the antimicrobial peptide, which will also help to improve the activity of the antimicrobial peptide.

The standard Fmoc solid-phase chemical synthesis scheme is adopted, that is, the two reactions of deprotection and activation cross-linking are repeatedly cycled to finally synthesize the peptide. The crude polypeptide synthesized is purified on a high-pressure liquid chromatography (HPLC), and a C18 preparative column is used for purification, and the purity reaches more than 95%. The purified product was analyzed by reversed-phase liquid chromatography (RP-HPLC) and mass spectrometry (MS) to identify its molecular weight, charge distribution, purity and quality.

The secondary structure of described 4 antimicrobial peptides:

SEQ ID NO.1:

SEQ ID NO.2:

SEQ ID NO.3:

SEQ ID NO.4:

Application of antimicrobial peptides:

The application of said SEQ ID NO.1 in the medicine of inhibiting MRSA, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa.

The application of said SEQ ID NO.2 in the medicine of inhibiting MRSA, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa.

The application of said SEQ ID NO.3 in the medicine of inhibiting escherichia coli and Pseudomonas aeruginosa.

The application of said SEQ ID NO.4 in the medicine of inhibiting enterococcus faecalis and escherichia coli.

The beneficial effect of technology described in this application is:

The four antimicrobial peptides of the present invention have shorter peptide chains, and each peptide chain has no more than 4 amino acid residues, which makes the synthesis easy; the charge is high, and each designed polypeptide has at least two positive charges; the amphiphilicity is strong, Ensure that it has low hemolytic toxicity; more dominant amino acids; and the end of the peptide chain is protected by ethylaniline with good flexibility and strong hydrophobicity. The four antimicrobial peptides have good structural specificity, strong resistance to enzymatic hydrolysis in vivo, clear mechanism of action, less toxic and side effects, easy chemical synthesis, and potential for drug production.

Description of drawings

Fig. 1 is the HPLC figure of SEQ ID NO.1 in embodiment 2.

Fig. 2 is the MS figure of SEQ ID NO.1 in embodiment 2.

Fig. 3 is the HPLC figure of SEQ ID NO.2 in embodiment 2.

Fig. 4 is the MS figure of SEQ ID NO.2 in embodiment 2.

Fig. 5 is the HPLC figure of SEQ ID NO.3 in embodiment 2.

Fig. 6 is the MS figure of SEQ ID NO.3 in embodiment 2.

Fig. 7 is the HPLC figure of SEQ ID NO.4 in embodiment 2.

Fig. 8 is the MS figure of SEQ ID NO.4 in embodiment 2.

Detailed ways

The above content of the invention of the present invention will be further described in detail below in conjunction with specific embodiments.

However, it should not be construed that the scope of the above-mentioned subject matter of the present invention is limited to the following examples. Without departing from the above-mentioned technical idea of the present invention, various replacements and changes made according to common technical knowledge and customary means in this field shall be included in the scope of the present invention.

Example 1:

This embodiment is the design and synthesis of the antimicrobial peptide:

1. Perform a primary sequence comparison analysis on the antimicrobial peptides in the existing antimicrobial peptide database (APD2) to determine the sequence pattern of the cationic antimicrobial peptides;

2. Carry out quantitative structure-activity relationship research on existing cationic antimicrobial peptides, and determine the dominant amino acids of the core sequence and their chemical modification methods;

3. Based on sequence patterns, amino acids at dominant sites and their chemical modifications, virtual generation of antimicrobial peptide libraries with diverse structures;

4. Screen and synthesize based on the results of structure-activity relationship analysis;

5. Screen out the best designed antibacterial peptides through antibacterial activity, cytotoxicity, and plasma stability tests.

The above SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 were synthesized in Shanghai Jier Biochemical Co., Ltd., and SEQ ID NO.4 was synthesized in Shanghai Lyon Chemical Co., Ltd., all using the standard Fmoc solid-phase chemical synthesis scheme , the two reactions of deprotection and activation of cross-linking are repeatedly cycled and finally the peptide synthesis will be prepared.

Purification, analysis and identification of the obtained polypeptide: the crude polypeptide synthesized in the above steps was purified on a Delta600 high-pressure liquid chromatograph (HPLC), and a C18 preparative column was used for purification, and the purity reached more than 95%.

Example 2:

This embodiment is the confirmation of the structure of the antimicrobial peptide:

The prepared designed peptide was analyzed by mass spectrometry (MS), and the error between the calculated molecular weight and the theoretical molecular weight of the polypeptide sequence shown in the mass spectrogram was at most about 1/‰, which proved that the prepared peptide was the designed antibacterial peptide. Its purity is above 95% (see Table 1) through reverse-phase high-performance liquid chromatography detection, and it is identified as a qualified peptide mimic product for future use.

Table 1 Mass Spectrometry Analysis and Purity of Synthesized Peptides

The HPLC and MS data are shown in Figures 1-8.

Example 3:

The present embodiment is the active experiment of described antimicrobial peptide:

Experimental steps:

a. Activation of bacteria: Prepare 2 bottles of 150ml solid LB culture solution, 4 bottles of 100ml liquid LB culture solution, and sterilize equipment such as petri dishes with high temperature steam for 30min. Pour the solid LB culture solution into 4 plates, and use the inoculation loop to pick from the Enterococcus faecalis, Staphylococcus aureus, MRSA, Pseudomonas aeruginosa, and Escherichia coli stored at -20°C, and place them on the plates respectively. To underline, to mark. Cultivate upside down overnight (12-16h) in a constant temperature incubator at 37°C.

b. Cultivate bacteria: pick a single colony into 100ml liquid LB culture medium, put it at 37°C, 170 rpm, and culture it on a shaking table overnight (12-16h).

c. Bacteriostatic activity test: Enterococcus faecalis, Staphylococcus aureus, MRSA, Pseudomonas aeruginosa and Escherichia coli were respectively prepared into 10 ⁵ -10 ⁶ cfu/ml bacterial suspension, and the bacterial solution was 0.2ml/ The amount of the plate (diameter of the plate is 150mm) is evenly spread on 40ml solid LB medium; after the medium is completely solidified, punch 10 holes/plate with a diameter of 8mm, and mark the positive control (gentamycin) and the negative control (deionized water), and 4 designed peptoids. The antimicrobial peptides with a purity of more than 95% in the above synthetic experiments were dissolved in dimethylsulfoxide (DMSO) and 0.01mol/L phosphate buffered saline (PBS) to a concentration of 1mg/ml (DMSO concentration<5‰) for use in Experiments for the verification of antibacterial activity. Add 50 μl 1mg/ml gentamicin to the positive control wells; add 50 μl deionized water to the negative control wells; add 50 μl 1 mg/ml peptidomimetic solution to the 4 designed peptidomimetic wells respectively. After standing at 4°C for 3 hours, culture in a constant temperature incubator at 37°C. After 7 hours, observe the size of the inhibition zone with the naked eye and measure the diameter of the inhibition zone. Each peptidomimetic was independently carried out 3 experiments under each bacterial species.

Among them, the synthetic peptides SEQ ID NO.1 and SEQ ID NO.2 have obvious antibacterial activity against MRSA, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa, and SEQ ID NO.3 has obvious antibacterial activity against Escherichia coli and aeruginosa Pseudomonas has obvious inhibitory effect, and SEQ ID NO.4 has obvious inhibition zone to Enterococcus faecalis and Escherichia coli.

Example 4:

The present embodiment is the bacteriostatic activity stability experiment of described antimicrobial peptide:

Continue to place the plate in the experiment of Example 3 in a constant temperature incubator at 37°C for continuous cultivation for one week, observe once every 12 hours, record the change of the inhibition zone within 7 days, and determine the duration of sterilization.

Experiments showed that the inhibition zone of the peptidomimetic SEQ ID NO.1 and the positive control gentamicin shrunk after 10 hours of culture, forming concentric circles with lighter color. This is likely to be due to the gradual decrease in the concentration of the drug solution as it diffuses outward. When the inhibition zone shrinks to 16mm size, it will not change in the next 3 days. These changes indicate that although the designed peptoid SEQ ID NO.1 has strong bactericidal ability, it cannot inhibit bacterial growth when the concentration is low, and the bactericidal effect is relatively stable and can last for a long time in the effective bactericidal concentration.

Although the diameter of the inhibition zone of the peptidomimetic SEQ ID NO.2 is small, it does not change after continuous culture, indicating that its bactericidal stability is better, or its low penetration ability makes it maintain a higher drug in a certain range Concentration, so as to be able to continue to sterilize.

The inhibition zone of peptidomimetic SEQ ID NO.3 and SEQ ID NO.4 became irregular after one day, and the inhibition zone also shrunk to varying degrees, but the process was relatively slow, and it did not change after shrinking inwardly by 1mm. It shows that the sterilization persistence is better.

Example 5:

The present embodiment is the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) determination experiment of described antimicrobial peptide:

Determined by the micro-fold dilution method.

The minimum inhibitory concentration MIC (Minimal inhibitory concentration) is the minimum concentration that can inhibit the growth of bacteria. In the antibacterial drug screening experiment, the antibacterial activity index of the drug is objectively verified, and the MIC ₅₀ is often measured, which is the concentration of the drug that can inhibit 50% of the bacterial growth. MIC ₅₀ is calculated by the bacteriostatic rate of a set of concentration gradients according to the calculation tool. Inhibition rate = [1-(experimental group-positive control)/(growth control-positive control)]×%. The minimum bactericidal concentration MBC (Minimal bactericidal concentration) is the lowest concentration of peptoids that can completely inhibit the growth of any residual colonies.

Experimental steps:

a. Culture Enterococcus faecalis, Staphylococcus aureus, MRSA, Escherichia coli, and Pseudomonas aeruginosa on a sterilized LB medium plate overnight, pick a single colony and inoculate it into the LB culture medium of a sterilized Erlenmeyer flask, Cultivate at 37°C and 170r/min for 12h.

b. Dilute the cultured bacterial solution at a ratio of 1:1000 (to make the number of bacteria reach 10 ⁵ -10 ⁶ cfu/ml). The peptidomimetic solution to be tested with antibacterial activity was serially diluted by 2 times and added to a 96-well microculture plate. The first and 12th wells were left blank (to avoid marginal effects resulting in inaccurate data). In addition to adding 160 μl of diluted bacterial solution to well 2, add 100 μl to each well from 3 to 9, then add 40 μl of antibacterial peptidomimetic stock solution (concentration 1280 μg/ml) to well 2, mix and aspirate 100 μl into the second well, and dilute in turn To the ninth well, discard 100 μL. The final mass concentrations of the antimicrobial peptides to be tested are respectively:

Take the tested peptidomimetic with a concentration of 1280 μg/mL and dilute it to the following concentration (μg/ml):

The growth control in the 10th well, and the positive control (gentamycin) in the 11th well were mixed with 160 μl of bacterial solution and 40 μl of antibiotic solution of the same concentration, and 100 μl was discarded. After culturing at 37°C for 16 hours, use an ELISA microplate reader to measure the OD value at 600nm, and calculate the effects of the peptoids on Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus and feces _MIC50 for Enterococci.

c. Determination of peptidomimetic MIC ₅₀ was calculated according to the minimum peptoid concentration at which the turbidity of control wells (11 wells) was lower than 50%. The _MIC50 of each bacterium liquid was independently tested 3 times for each peptoid, and the average value was taken.

d. Take 10 μl from the contents of the lowest plate well without bacterial growth and spread it on the LB agar plate, incubate at 37°C for 18 hours, and observe whether there is colony growth, so as to determine the minimum bactericidal concentration (MBC) of the peptidomimetic.

The 4 peptoids were used to measure MIC and MBC of Staphylococcus aureus, MRSA, Escherichia coli, Pseudomonas aeruginosa, and Enterococcus faecalis in a microplate reader at 600nm. The MIC of each bacterium was tested independently for 3 times for each peptidomimetic, and the average value was taken to determine the MIC and MBC of the peptidomimetic, see Table 2 and Table 3. It can be seen from the table that the antibacterial ability of the designed peptide has a good effect whether it is a gram-positive bacterium or a gram-negative bacterium. In theory, the concentration of BMC should be in the upper well where MIC50 appears, but due to the low concentration gradient range set in this experiment, the BMC of peptides with higher minimum inhibitory concentrations of SEQ ID NO.3 and SEQ ID NO.4 did not fall within the concentration range. It is not meaningful to further measure the BMC of these peptides with weak antibacterial activity, so it will not be considered.

Table 2 Comparison of MIC50 of 4 kinds of peptoids against different bacteria

Table 3 Comparison of minimum bactericidal concentration (MBC) of 4 kinds of peptoids against different bacteria

"—" indicates that the bacteria were not completely killed at the highest concentration.

Embodiment 6:

Present embodiment is the hemolytic toxicity experiment of described antimicrobial peptide:

In the safety evaluation of drugs, the determination of hemolysis is an important index. If hemolysis occurs, it proves that the drug has a serious toxic reaction to the body. Red blood cells have a certain resistance to hypotonic solutions, which is called osmotic fragility of red blood cells. When the concentration of NaCl solution is as low as 0.42% to 0.46%, some red blood cells will rupture due to excessive expansion, and hemoglobin will escape from the red blood cells. This phenomenon is called hemolysis. When the concentration of NaCl solution is as low as 0.32% to 0.34%, all red blood cells are hemolyzed. Whether an antimicrobial peptide has an inhibitory effect on normal human cells, that is, whether it has cytotoxicity, is one of the key factors that determine whether an antimicrobial peptide can be developed into a drug.

Experimental steps:

a. Centrifuge sheep blood at 3000rpm/min for 10min and wash with PBS buffer (10mM 138mM NaCl and 2.7mM KCL, PBSpH 7.4) for 3 times, discard the supernatant, leaving only red blood cells.

b. Dilute 0.1ml of erythrocyte fluid in 9.9ml of PBS (final concentration of erythrocytes is 1%) 200μl and incubate at 37℃ with 200μl of various concentrations of antimicrobial peptides of 640, 320, 160, 80, 40, 20 (μg/ml) After 1 hour, centrifuge at 4000rpm/min for 5 minutes, transfer the suspension to a 96-well ELISA plate and detect the absorbance of the suspension at a wavelength of 414nm, and calculate the hemolysis rate and LD50 value of each well. 0.01mol/L PBS was used as a negative control, and 0.1% Triton-X 100 (polyethylene glycol octylphenyl ether) was used as a positive control. Hemolysis rate (%)=(absorbance of test tube-absorbance of negative control tube)/(absorbance of positive control tube-absorbance of negative control tube)×100%[62]. The smaller the hemolytic rate of the antimicrobial peptide, the smaller the hemolytic toxicity of the antimicrobial peptide.

Table 4 Hemolysis rate of 4 kinds of antimicrobial peptides

Embodiment 7:

The present embodiment is the plasma stability experiment of described antimicrobial peptide:

The stability of antimicrobial peptides in plasma has an important influence on their actual antibacterial ability. If the antimicrobial peptide is too easy to be enzymatically hydrolyzed, it will not be druggable. In this experiment, reversed-phase high-performance liquid chromatography was used to detect its residual concentration after reacting with plasma in vitro for different times, and its half-life in plasma was calculated according to the percentage of peak area.

a. Prepare the 4 peptides with deionized water respectively to a 1 mg/ml solution; dilute the inactivated plasma to a concentration of 25% for later use.

b. Take 1.5ml sterile dry EP tubes and label them as A0, A1, A2, A3, A4, A5, A6 in turn, A0 represents the blank control, that is, add 200μl prepared peptide solution and 200μl deionized water, then add 600μl acetonitrile . Add 200 μl of prepared peptide solution and 200 μl of spare plasma to each tube of A1-A6, so that the concentration ratio of drug to plasma is 1:1. A1-A6 represent the different reaction time points of the experimental groups, which are 0.5, 1, 1.5, 2, 2.5, and 3 (h) in sequence. Put the EP tubes of the 6 experimental groups except A0 into a 37°C water bath for suspension heating. After timing for 5 minutes, take out A1, immediately add 600 μl (to make the total volume 1ml) acetonitrile to terminate the reaction, and then put it in a refrigerator at 4°C for 20 minutes to complete the reaction. Take out A2 after 10 minutes, and repeat the above operations in turn until A6 is taken out at the 30th minute, add acetonitrile to stop the reaction and stand at 4°C for 20 minutes, then centrifuge at 12,000r/min for 15min, draw 500μl of the supernatant and use a high-performance liquid chromatography analyzer detection.

c. Chromatographic analysis adopts Agilent1220 high-performance liquid chromatography and Welch Ultimate XB-C18 chromatographic column, mobile phase: A liquid: DDW+0.1% TFA, B liquid: acetonitrile+0.1% TFA; flow rate: 1.0ml/min; detection Wave 220nm; column temperature: room temperature; gradient method; 0min B solution 15%, 30min B solution 45%, 30.1min B solution 100%, 31min B solution 100%, 37min Stop.

d. Integrate the peak area at the same retention time as the blank control, which is the peak area at each time point drug concentration. In the HPLC liquid chromatography analysis, under the same conditions, the concentration is proportional to the peak area, so we set The peak area of the blank control is 100%, and the ratio of the peak area of the remaining experimental groups to the peak area of the blank control is the percentage of its concentration. According to this method, the concentration of each time point of the experimental group was calculated. The above experiments were repeated three times independently, and the average value was calculated.

The results showed that the plasma stability of the peptidomimetic designed and synthesized in this project was greatly improved compared with the natural amino acid antibacterial peptide. It is proved that the non-natural modification of antimicrobial peptides can greatly improve its plasma stability.

In summary, the four antimicrobial peptides described in the present invention have good structural specificity, strong resistance to enzymatic hydrolysis in vivo, clear mechanism of action, less toxic and side effects, easy chemical synthesis, and potential for drug production.

sequence listing

<110> Chongqing University of Technology

<120>A non-protein amino acid antimicrobial peptide and its application

<160>4

<170>PatentIn Version 3.1

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<213> Artificial design

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<222>(1)...(4)

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R FPA R FPA EtBn

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<213> Artificial design

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R BTF R BTF EtBn

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<213> Artificial design

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R FPA R EtBn

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<213> Artificial design

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R Nag R EtBn

1

Claims (2)

1. a non-protein amino acid antibacterial peptide, is characterized in that: the sequence of described antibacterial peptide is as shown in SEQ ID NO.3 or SEQ ID NO.4:

SEQ ID NO.3:R-FPA-R-EtBn；

SEQ ID NO.4:R-Nag-R-EtBn；

Wherein R is arginine, and FPA is: nag is: etBn is ethylbenzene amination protecting group.

2. the application of a non-protein amino acid antibacterial peptide according to claim 1, it is characterized in that: by described non-protein amino acid antibacterial peptide for the preparation of antibacterial medicines, described antibacterial medicines can suppress at least one in intestinal bacteria, enterococcus faecalis and Pseudomonas aeruginosa.